Mammalian cells are usually cultured in glass or plastic vessels, either in suspension or as an attached layer, completely surrounded by culture media. Pseudopodia protrusion is a prominent feature of mammalian cells both invitro and invivo. Through their surfaces, cells can interact and sense the changes in the outside world. In studies of model membrane systems, steps in the fusion of membrane have been identified. In order for the fusion process to take place, certain requirements have to be fulfilled. Initially the membranes have to be close enough to make contact. This is generally prevented by large membrane glycoproteins which sterilly hinder the membranes preventing a close enough contact to allow fusion. Because of the charged surface of the membranes, they may repel, thereby preventing any closer contact. Therefore, there must be some mechanism within the cell to control which membrane will undergo fusion. This may be regulated by specific fusion proteins similar to the ones isolated from the influenzia and sendia virus.
The use of chambers for cell culture and chemotaxis assay is known in the prior art.
U.S. Pat. No. 3,821,087 discloses an apparatus having semi-permeable tube shaped membranes allowing the growth of cells invitro. Cells are allowed to settle on the outside surfaces of the membranes in a nutrient medium environment while the membranes are continuously profused with oxygenated nutrient medium flowing through the membranes. The nutrient substances diffuse from the perfusing medium through the membrane wall and into the cells, while cell products diffuse from the cell through the membrane wall into the perfusate from which the cell's products may be recovered.
U.S. Pat. Nos. 4,087,327 and 4,201,845 are directed to a cell culture reactor used in the growth of cells invitro which employs elongate selectively permeable hollow fibers arranged in a shallow layer configuration as a matrix for cell attachment on the outer surface of the fibers. The flow path of the culture medium is directed by a distributor plate which serves as a manifold to provide uniform distribution of the medium through the fibers and a flow path which is upward and transverse to the plane of the elongate axis of the fibers.
U.S. Pat. No. 4,228,243 discloses an apparatus and method for propagating tissue cultures in a plurality of stacked frames and plates which are clamped together. The plates are stacked on resilient gaskets which are provided to stop leakage between the uppermost dish and coverplates and overflow passages are provided in the plates through which the media, cell suspensions and gases can pass.
U.S. Pat. No. 4,299,920 discloses a receptacle for cell cultures constructed of a base plate with a wall assembly defining a plurality of chambers detachably, adhesively joined to the base plate. The portion of the wall assembly adjacent to the base plate consists of a noncytotoxic elastomeric synthetic material which adheres to the base plate in a liquid-tight manner. On completion of cultivation of the cells the base plate can be manually separated from the wall section.
U.S. Pat. No. 4,304,865 discloses an apparatus having a harvester plate with a plurality of wells recessed into one surface. A disc-shaped sheet of filter paper placed over the top surface of the harvester plate and a modified Terasaki plate with an outer wall section and conically shaped wells recessed into its surface is mounted over the filter paper and harvester plate. When the plate is maintained in an upside down orientation the material is transferred directly to the filter paper disc.
U.S. Pat. No. 4,324,859 discloses a microbiological container with a substrate having insertable elements. The lower edge of each element forms a sealed relationship with the bottom of the container forming separate regions with isolated substrate portions. A cover can be placed on the insertable element in order to cover the respective isolated section.
U.S. Pat. No. 4,326,028 discloses a cylindrical dish with two cylindrical compartments divided by a perforated jell support structure with a temporary seal. After the microorganisms are grown in compartment A, the dish is inverted, the seal removed, and a second nutriate medium poured into compartment B.
U.S. Pat. No. 4,634,676 discloses a replica plating device comprising a container which holds cells and a skirted press member which fits into the container. Attached to the exterior bottom surface of the bottom wall of the press member is a layer of compressible material such as soft expanded polyurethane or hard polyurethane foam. The compressible material serves to cushion the pressing effect of the replica plating device against the cells and culture medium during use.
U.S. Pat. No. 4,661,458 discloses a device used for the culture of cells incorporating several modules of different functions for regulating the cell growth environment, providing a suitable cell growth substrate or separating the desired product from interfering substances. Each module is constructed of a series of membranes separated by a solid support material which is channeled to provide a series of parallel capillaries for the flow of fluids.
U.S. Pat. No. 4,720,462 discloses a culture tube divided into five sealed chambers with cell impermeable hydrophilic tubes extending from one chamber to another chamber.
A cell permeable filter tube extends along the initial horizontal axis of the straight culture tube from one end of the culture chamber to the other. The filter tube has an average porosity over 100 microns, preferably between about 100 and 500 microns such that under normal operating conditions of cell sloughage the tube does not occlude. Cells are forced under pressure into the tube and through the wall of the cell permeable tube.
U.S. Pat. No. 4,748,124 discloses a closed cell culture device using spaced sheets of gas permeable, liquid impermeable material with a third sheet sandwiched between the first and second spaced sheets which is selectively permeable to a class of molecules having a molecular weight ranging from 8,000 to 15,000 molecules. All of the sheets are held in a housing chamber formed by four concentric ring members, the first and third sheets define a first closed compartment and the second and third sheets define a second enclosed compartment with each compartment having an access port. The rings are sealed together in compression by attachment screws.
The use of a small diameter pore filter which is small enough to prevent whole cell migration in the chemotaxis assay allows the cells to first extend their pseudopodia to a lower compartment while the cell nuclei remain in the upper compartment. Consequently the separation between the two compartments by mono layered cells anchored to the filter through their extended pseudopodia permits the collection of extended pseudopodia as well as substances secreted into the lower walls. Thus, depending on the pore size of the filters, the pseudopodia chamber can be used for the study of either whole cell migration or cell pseudopodia protrusion. Although cell pseudopodia is not new to researchers studying cell movement, no one has previously been able to isolate these cellular processes. Furthermore, in order to study the morphological changes involved in the intermediate steps between triggering the chemotaxtic signal and the end result i.e. cell locomotion, one had to rely on a complicated technique such as time lapse cinemotagraphy. The results of such cinemotagraphy are difficult to interpret as well as uncertain.
One prior art assay for enutorphoril chemotaxis which ha been widely used for studying cell movement is the Boyden chamber assay which is shown in FIGS. 1 and 1A. The Boyden chamber is composed of two parts which constitute an upper and a blind lower well. Chemo attractants are added to the lower well before placing the membrane which separates the lower from the upper well. In this assay the conditions in the lower well cannot be changed once the experiment has been started. A second prior art chamber which has been used for chemotaxis assay is shown in FIGS. 2 and 2A. This chamber is composed of a bottom plate with a single or multiple blind wells, a filter and a top plate with matching diameter wells which is attached to the bottom plates by a series of bolts. In contradistinction to this device, the newly designed inventive chamber provides free access to both the upper as well as the lower wells throughout the experiment. This permits the study of isolated or intact pseudopodial protrusions independent from their cell bodies. Moreover, since the cell monolayer can physically separate the two compartments in the new chamber, one can further study the changes in cell responses to different legans after changing the conditions in either the upper or lower wells or both.